Lung cancer is responsible for the most cancer-related deaths worldwide. This proposal aims at dissecting the signaling pathways perturbed in human lung cancer, with a focus on the mechanisms of DOK-mediated lung tumor suppression. DOK (downstream of tyrosine kinase) family proteins are signaling proteins that modulate tyrosine kinase signaling. We recently identified DOK-1, DOK-2, and DOK-3 as lung tumor suppressors, and we further hypothesize that they are negative regulators of oncogenic EGFR-RAS signaling. In our preliminary analysis, we find that Dok-1, Dok-2, and Dok-3 single, double, and triple knockout mice develop lung adenocarcinoma with a penetrance and latency dependent on the number of lost Dok alleles. Tumors from Dok null mice exhibit Akt and Erk activation, similar to models of EGFR- or KRAS-driven tumorigenesis. In human non-small cell lung cancer (NSCLC), we observe frequent genomic loss of DOK-2 with a concomitant reduction of DOK-2 mRNA expression. Strikingly, genomic loss of DOK-2 is strongly associated with EGFR mutation status. We therefore hypothesize that DOK-2 opposes lung tumorigenesis initiated by oncogenic EGFR and RAS. Moreover, we further hypothesize that variant alleles of DOK-2 (such as DOK-2L138S) could underlie lung cancer susceptibility. The purpose of this proposal is to determine the mechanisms of DOK-mediated lung tumor suppression vis--vis the EGFR-RAS pathway and to ascertain the relevance of variant DOK-2 alleles and non-pulmonary cellular compartments to the pathogenesis of DOK-null lung cancer with the following specific aims: (1) To define the role of DOK-2 in EGFR- and KRAS-mutant lung cancers. To this end, we will utilize already existing EGFR and KRAS bitransgenic and Dok-2-/- mouse models to determine if Dok-2 opposes mutant EGFR or KRAS-driven lung cancer in vivo. In a preliminary analysis, we find that Dok-2 does indeed oppose lung tumorigenesis initiated by KRASG12D. (2) To define the role of DOK-2L138S in lung cancer susceptibility. The DOK-2L138S allele is present both as a somatic mutation found in human lung cancer and as a naturally occurring germline polymorphism. In vitro analysis indicates that DOK-2L138S is a loss-of-function mutant that is defective at suppressing EGF-induced RAS and ERK activity. To determine if this allele contributes to lung cancer susceptibility, we will perform a case/control study of human lung cancer, and, in parallel, generate a Dok-2L138S knock-in mouse model. (3) Evaluate the contributions of cell autonomous and non-cell autonomous mechanisms to tumor formation in Dok mutant mice. The relevance of the Dok TKO hematopoietic phenotype will be determined using a reciprocal bone marrow transplant using wild-type and TKO animals. Furthermore, generation of a lung-specific conditional Dok-2 knockout mouse model will allow unequivocal determination of the contribution of cell autonomous mechanisms to the Dok-2 knockout lung phenotype.